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eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation.

Brown K, Doss MX, Legros S, Artus J, Hadjantonakis AK, Foley AC - PLoS ONE (2010)

Bottom Line: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation.Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts.These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Greenberg Division of Cardiology, Weill Cornell Medical College, New York, New York, United States of America.

ABSTRACT

Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/principal findings: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/significance: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

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Addition of XEN-CM and PYS2-CM, but not END2-CM increases the amount of cardiomyocytes that form in culture and increases expression of cardiac markers as assessed by qRT-PCR.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other two cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 4–6 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on day 10 and 13 after addition of CM on days 4–6. (*indicates a p<0.05). G. qRT-PCR data showing expression of cardiac markers at day 7 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
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pone-0013446-g002: Addition of XEN-CM and PYS2-CM, but not END2-CM increases the amount of cardiomyocytes that form in culture and increases expression of cardiac markers as assessed by qRT-PCR.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other two cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 4–6 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on day 10 and 13 after addition of CM on days 4–6. (*indicates a p<0.05). G. qRT-PCR data showing expression of cardiac markers at day 7 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).

Mentions: We noted that while all three CMs increased the percentage of EBs that formed beating areas, beating areas remained relatively small in response to END2-CM. In contrast addition of PYS2-CM and XEN-CM increased both the number and size of the beating areas (Compare the difference Figure 2A and 2B to the differences between Figure 2C to 2 D, E). To further assess this effect, we performed flow cytometry on cells isolated from day 10 and day 13 EBs using the MHCα::GFP reporter to identify cells possessing a cardiac fate (Figure 2F). GFP-positive cells were analyzed and assessed as a percentage of total cells counted. Consistent with our data showing an increase in the number of EBs that beat, addition of XEN-CM or PYS2-CM from days 4–6 increased the percentage of GFP-positive cardiomyocytes on day 10. However by day 13, only EBs treated with XEN-CM continued to show a statistically significant increase in the number of cardiomyocytes. By contrast, treatment of EBs with END2-CM had no effect on the number of GFP-positive cells present at day 10 or day 13. Thus, while END2-CM increased the likelihood that an EB would form beating areas, it appeared that either cardiac progenitors did not expand after addition of END2-CM or that they failed to further supplement endogenous cardiogenic signals that arose from visceral endoderm formed by the EB. This finding was consistent with previous data demonstrating that CM from END2 cells increased cardiac differentiation of human ES cells [22], [23] but that co-culture was required to enhance cardiac differentiation from mouse ES cells [31].


eXtraembryonic ENdoderm (XEN) stem cells produce factors that activate heart formation.

Brown K, Doss MX, Legros S, Artus J, Hadjantonakis AK, Foley AC - PLoS ONE (2010)

Addition of XEN-CM and PYS2-CM, but not END2-CM increases the amount of cardiomyocytes that form in culture and increases expression of cardiac markers as assessed by qRT-PCR.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other two cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 4–6 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on day 10 and 13 after addition of CM on days 4–6. (*indicates a p<0.05). G. qRT-PCR data showing expression of cardiac markers at day 7 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958120&req=5

pone-0013446-g002: Addition of XEN-CM and PYS2-CM, but not END2-CM increases the amount of cardiomyocytes that form in culture and increases expression of cardiac markers as assessed by qRT-PCR.A, C. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes in control EBs. A separate control is included for END2 cells since they are grown in different medium from the other two cell lines. B, D, E. Merge of bright field and pseudo-colored fluorescence images to show distribution of cardiomyocytes after treatment on days 4–6 with (B) END2, (D) PYS2 and (E) XEN-CM. F. Summary of flow cytometry data showing the fold change in the number of MHCα::GFP (+) cells on day 10 and 13 after addition of CM on days 4–6. (*indicates a p<0.05). G. qRT-PCR data showing expression of cardiac markers at day 7 after treatment of EBs with CM on days 4–6. (* indicates p<0.05).
Mentions: We noted that while all three CMs increased the percentage of EBs that formed beating areas, beating areas remained relatively small in response to END2-CM. In contrast addition of PYS2-CM and XEN-CM increased both the number and size of the beating areas (Compare the difference Figure 2A and 2B to the differences between Figure 2C to 2 D, E). To further assess this effect, we performed flow cytometry on cells isolated from day 10 and day 13 EBs using the MHCα::GFP reporter to identify cells possessing a cardiac fate (Figure 2F). GFP-positive cells were analyzed and assessed as a percentage of total cells counted. Consistent with our data showing an increase in the number of EBs that beat, addition of XEN-CM or PYS2-CM from days 4–6 increased the percentage of GFP-positive cardiomyocytes on day 10. However by day 13, only EBs treated with XEN-CM continued to show a statistically significant increase in the number of cardiomyocytes. By contrast, treatment of EBs with END2-CM had no effect on the number of GFP-positive cells present at day 10 or day 13. Thus, while END2-CM increased the likelihood that an EB would form beating areas, it appeared that either cardiac progenitors did not expand after addition of END2-CM or that they failed to further supplement endogenous cardiogenic signals that arose from visceral endoderm formed by the EB. This finding was consistent with previous data demonstrating that CM from END2 cells increased cardiac differentiation of human ES cells [22], [23] but that co-culture was required to enhance cardiac differentiation from mouse ES cells [31].

Bottom Line: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation.Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts.These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

View Article: PubMed Central - PubMed

Affiliation: Greenberg Division of Cardiology, Weill Cornell Medical College, New York, New York, United States of America.

ABSTRACT

Background: Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.

Methodology/principal findings: Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.

Conclusions/significance: These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm.

Show MeSH